CORRELATION BETWEEN SPECIFIC GENETIC MUTATIONS AND COLISTIN - - PowerPoint PPT Presentation

AN INVESTIGATION INTO THE CORRELATION BETWEEN SPECIFIC GENETIC MUTATIONS AND COLISTIN RESISTANCE IN ESCHERICHIA COLI

Leisha McGrath – B.Sc.(Hons.) in Medical Science Galway-Mayo Institute of Technology University College Dublin-Centre for Food Safety

BACKGROUND OF COLISTIN

• Last line of defence for treating multi-drug resistant Gram

negative bacterial infections

• Cationic polypeptide with a fatty acid chain attached
• Targets the lipopolysaccharide molecule
• Use in human medicine becoming more common

COLISTIN IN MEDICINE

• Manufactured as colistin sulfate or colistimethate sodium
• Colistin sulfate used in bowel decontamination and treating

surface infections

• Colistimethate sodium used to treat MDR Pseudomonas

aeruginosa infections in cystic fibrosis patients

• Associated with nephrotoxicity and neurotoxicity

CHROMOSOMAL COLISTIN RESISTANCE

Low [Mg2+] High [Fe3+]

Figure 1: Colistin resistance mechanisms (1)

PROJECT METHODOLOGIES

• 1. Microbroth Dilution to investigate antibiotic

minimum inhibitory concentrations

• 2. Lambda Red Recombination for deleting

phoP from E. coli ATCC 25922 genome

• 1. MICROBROTH DILUTION

Antibiotic conc. (Mg/l) S.C. G.C. 0.0156 0.0313 0.0625 0.125 0.25 0.5 1 2 4 8 Well No. 1 2 3 4 5 6 7 8 9 10 11 12 M6 M73 M33 M52 M4 M44 Control ATCC 25922

Table 1: Schematic table of microbroth dilution method

• 2. LAMBDA RED RECOMBINATION

Figure 2: Schematic demonstrating Lambda Red Recombination (2)

Isolate mcr-1 Status of Isolate Colistin MIC Interpretation

M6 mcr-1 negative RESISTANT M73 mcr-1 negative RESISTANT M33 mcr-1 positive LOW LEVEL RESISTANCE M52 mcr-1 positive SENSITIVE M4 mcr-1 negative SENSITIVE M44 mcr-1 negative SENSITIVE Control ATCC 25922 mcr-1 negative SENSITIVE

Table 2: mcr-1 status and colistin MIC interpretation of E. coli test isolates and control strain

VERIFICATION OF phoP DELETION

Figure 4: Electrophoresis demonstrating the presence of kanamycin resistance cassette in Well 2 and phoP in Wells 2 and 4

VERIFICATION OF KRC DELETION

Figure 5: Electrophoresis graph demonstrating the successful deletion of the KRC from mutant E. coli ATCC 25922

Colistin MIC (mg/l) Interpretation Wild type E. coli ATCC 25922 0.125 SENSITIVE Mutant E. coli ATCC 25922 0.25 SENSITIVE Table 3: Colistin MIC of wild type E. coli ATCC control strain and mutant control strain

CONCLUSION

• One fold increase in MIC value is significant
• Demonstrates other two-component systems compensate

for loss of phoP upon colistin exposure

• Also implies phoP mutation alone cannot mediate colistin

resistance

• Multiple mutations in two-component systems are required

for the development of a colistin resistant phenotype

FUTURE WORK

• Clone mcr-1 into:
• Susceptible E. coli control strain ATCC 25922
• Mutant E. coli ATCC 25922
• Investigate the true function of the mcr-1 gene – does it

actually confer colistin resistance or serve to augment colistin resistance mechanisms?

REFERENCES

• (1) Olaitan AO, Morand S, Rolain J-M. Mechanisms of

polymyxin resistance: acquired and intrinsic resistance in

• bacteria. Frontiers in Microbiology. 2014; 5: 634.
• (2) Datsenko KA, Wanner BL. One-step inactivation of

chromosomal genes in Escherichia coli K-12 using PCR

• products. Proceedings of the National Academy of

Sciences of the United States of America. 2000; 97(12): 6640 – 6645.

ACKNOWLEDGEMENTS

I would like to sincerely thank:

• Professor Séamus Fanning, Director of the UCD Centre for

Food Safety and Principle Investigator of this project, for his guidance and advice during this research project

• Evan Brennan and Dr. Shabarinath Srikumar, researchers in

UCD Centre for Food Safety for their help in both the theoretical and practical aspects of this project

• Dr. Debbie Corcoran, Galway-Mayo Institute of Technology

Microbiology lecturer, for organising this research project with UCD and for all the support and encouragement given throughout the duration of the project